PROJECT SUMMARY/ABSTRACT This is an application for an R21 NIH Exploratory/Developmental Research Grant Award. Our long-term goal is to determine how Mycobacterium tuberculosis (Mtb) disrupts the host immune response to establish infection in humans, and to use this information to develop better vaccines and therapies for tuberculosis (TB). In our prior work, we used proteomics to systematically define the interactions between secreted Mtb proteins and human proteins, and discovered a network of several hundred highly-specific interactions that potentially play important roles in Mtb pathogenesis and host defense. We began an initial genetic analysis of the interaction network, and discovered both a novel virulence factor LpqN, as well as its probable host target, the ubiquitin ligase CBL. The specific goal of this application is to use genetics to probe this biochemically-defined Mtb-host interactome, in order to identify functionally important interactions between Mtb and host proteins. In Aim 1 we will use bacterial genetics to disrupt the bacterial factors within the interactome, and will test these mutants for growth defects in both mice and isolated ex vivo macrophages. In Aim 2 we will use CRISPR technology to disrupt the host proteins that interact with those bacterial factors contributing to virulence. We anticipate that genetic analysis of the remaining interactome will identify several bacterial virulence factors, as well as additional host pathways playing important roles in Mtb immunity. These findings will pave the way for future studies that will seek to uncover the molecular mechanisms by which these host factors contribute to immunity, which may suggest ways that the factors can be manipulated for therapeutic benefit.